Method and detector for separation processes

ABSTRACT

The present invention comprises a method Of detecting and quantifying components separated by capillary electrophoresis or other techniques using thin capillaries in the separation. The method of the present invention comprises irradiating the whole or large parts of the capillary at short time intervals and detecting the fluorescence originating from the sample components, preferably by a CCD detector (charge coupled device). Thereby, an instantaneous image of the progress of the separation process is obtained. The present invention further comprises an apparatus for performing this detection.

In the bioscientific field there is a great need of being capable ofanalysing complex mixtures of biomolecules. Examples are the analysis ofmetabolites, proteins, antibodies and drugs in clinical analysis.Another example is the analysis of protein composition in thefermentation of proteins specified by genetic engineering.

A usual way of performing bioanalyses is to separate a sample into itsconstituents and then quantify the constituents. Conventional separationmethods are chromatography and electrophoresis. Electrophoresis has astrong position just for analysis. The reason is that the method ishigh-resolving, which among other things means that many components canbe analysed in one and the same run. In combination with suitabledetection methods for the separated substances, the method may also bemade very sensitive. On the other hand, the method is generally slow,typical separation times being in the range of 1-10 hours.

For the last couple of years a special form of electrophoresis hasattracted much attention, viz. capillary electrophoresis. Theelectrophoretic process takes place in thin capillaries of quartz(inside diameter about 0.005-0.1 mm). The thin capillaries make it easyto cool off the Joule heat that is generated during the process of theelectrophoresis. It is therefore possible to use very high fieldintensities in the electrophoretic separation, e.g. 300 V/cm. Since thehigh field strength gives a very rapid migration rate, the separationtimes become favourably short with capillary electrophoresis. In normalelectrophoresis, temperature gradients easily arise in the separationbed. Temperature gradients lead to a heavily impaired resolution of thesample components. This problem is eliminated by the capillaryelectrophoresis technique, where the thin capillaries are easy to cooland therefore do not lead to problematic temperature gradients.Consequently, also the resolving power is very good with capillaryelectrophoresis. An illustrative review article has been written by H.Carchon and E. Eggermont (International Chromatography Laboratory, Vol.6, pages 17-22, 1991).

The detection and quantification of mixtures separated by capillaryelectrophoresis are usually performed with a UV/Vis detector orfluorescence detector (a review of different detection principles forcapillary electrophoresis has been written by D. M. Goodall, D. K. Lloydand S. J. Williams in LC-GC, Vol. 8, No. 10, 1990, pages 788-799). Thesedetectors are always placed at one end of the capillary. Usually a lightbeam is made to pass perpendicularly through the capillary, and adetector measures the amount of light that has passed or the amount offluorescence that has been induced. The separated substances are therebydetected one after the other as they pass the detector. This means thatthe electrophoresis must continue until all the substances have passedthe detector. Often the number of substances in the sample is not known,and the electrophoretic run must therefore proceed considerably longerthan actually necessary in order to ensure that all the samplecomponents have really passed the detector. A limiting factor ofcapillary electrophoresis is thus seen here, viz. the serial detectionprinciple, i.e. the sample components being detected one after theother. If instead a detection principle is provided where the wholecapillary is examined momentarily by a special detector, a paralleldetention principle is obtained where all the components are detectedsimultaneously. The present invention discloses just such a principleand the construction of suitable apparatus.

With the present invention, a sample may be separated into components ina thin capillary. At short intervals during the separation, the whole,or at least a large part, of the capillary may be simultaneouslyirradiated and either the emitted or absorbed light may be detected,thus creating successive momentary images of the separation pattern. Thedetection of the light may be based on, but is not limited to,fluorescence and light scattering of the sample components.

The novel detection principle resides in monitoring the capillaryelectrophoresis by a detector which at short intervals examines thewhole capillary and registers where the sample components are in thecapillary. Simultaneously, the detector registers spectralcharacteristics of the sample components, which facilitates theiridentification. The data obtained are stored in a computer.

SUMMARY OF THE INVENTION

To explain the manner of function the following short apparatusdescription will give guidance (a more detailed description will begiven further on): The detector according to the present inventionconsists of a strong light source, e.g. a laser. The laser irradiatesthe whole capillary, preferably at short time intervals. Upon eachirradiation the spatially separated sample substances will fluoresce.The fluorescence emission from the capillary is focused via a lenssystem onto a CCD detector (charge coupled device), generating anelectronic image of the appearance of the capillary. A computer storesthis image. Between each irradiation time, the sample components move alittle, this movement being registered by the CCD detector. The computercontinuously transforms the stored data and can in real-time present theprogress of the separation on a display screen. This is a very greatadvantage. It may be seen directly on the computer screen when theseparation of the sample components is sufficiently good for them to bequantified with sufficient precision. Optionally the computer mayperform this judgement by itself. This means great savings of time,since the separation may be stopped much earlier than in the case of adetector placed at the end of the capillary. This is particularly thecase if the sample contains very slowly migrating components.

With the present invention it is possible to reverse the polarity of theelectrophoretic process when a sufficiently good separation has beenobtained and wash out the sample backwards to prepare the capillary forthe next sample. If the sample contains slowly migrating substances, itis easily understood that the invention leads to an improvement inefficiency (sample/hour) by a factor of 10.

Another advantage of the novel detector is that the operator/computerhas a superior control of the separation process. All separationprocesses with serial detection, including normal capillaryelectrophoresis, suffers from the problem that one cannot be sure thatall components have actually reached the detector when the separation isstopped. This problem is effectively solved by monitoring the wholeseparation capillary in accordance with the invention.

Fields which may particularly take advantage of the novel detector maybe envisaged. One of them is sequence analysis of genes, whereanalytical speed combined with reliable determination is very important.The sample, which has been generated via Sanger's method with controlledinterrupted replication, consists here of a mixture of oligonucleotidesof different lengths. The oligonucleotides are labelled with fluorescentdyes, e.g. Rhodamine 110, Rhodamine 6G, Tetramethyl Rhodamine andX-Rhodamine, which are well adapted to excitation by an argon laser (514nm). Very rapid separation cycles are possible here. As soon as asufficient separation has been obtained, the system may be regeneratedthrough pole reversal. Also much longer sequences than are normallypractical to work with will thereby be possible to handle.

Another very interesting field is the study of protein-ligandinteractions. If the protein has a higher migration rate than theligand, the ligand is first applied to the capillary. A while later theprotein is applied. The protein will now migrate into the slowlymigrating segment with ligand, and after still some time it will havepassed the ligand segment completely. In connection with the passage,interactions take place which will be reflected in the migration ratesof the components. The novel detector makes it possible to follow theseinteractions very accurately. By afterwards analysing the fronts arisenand their composition, very interesting information may be extractedconcerning equilibrium constants and kinetic rate constants.

DETAILED DESCRIPTION OF THE INVENTION

The above description of the invention relates to capillaryelectrophoresis. The same principal advantages apply to electrokineticchromatography, i.e. separation with capillary electrophoresis equipmentwhere the actual separation process, however, is not electrophoretic butchromatographic. Also separations by HPLC technique in thin glass orsilica columns may make use of the novel detection method.

There are a number of alternative embodiments of the invention,including various ways of connecting the light source (laser or otherlight source) to the capillary. One way is to irradiate the fiberperpendicularly to its longitudinal direction. Another way is to injectthe exciting light through the end surface of the capillary. This waymay be expected to give a particularly even and effective irradiationsimultaneously as the amount of stray light is minimized. A number oflaser types may be used as the excitation source, the choice beingdetermined inter alia by the desired excitation wavelength. A HeCd-laser(e.g. of Liconix make) will give suitable lines at 442 and 325 nm. Asmall argon ion laser (e.g. of Spectra Physics make) will giveblue-green lines, but also UV-light may be obtained around 350 nm. Apulsated nitrogen laser (e.g. of Laser Science Inc. make) emits at 337nm and may also be used to pump a dye laser for generating arbitraryvisible wavelengths.

The total fluorescence may without wavelength discrimination be imagedonto a linear detector (diode row) after the exciting laser light hasbeen suppressed by means of a sharp high-pass filter, suitably a colourfilter (e.g. of Schott make). Suitable diode rows for the purpose aremarketed by e.g. EG&G or Reticon. The diode row has often a length of 25mm and 1024 individual diodes, but is also available in longer designs.The lens system is selected such that the straight capillary is imagedwith correct reduction onto the diode row in such a way that each diodecorresponds to a certain position along the capillary. The spatiallyresolved detector signal may be read to a computer via a control unit.If a pulsated excitation source is used (e.g. a nitrogen laser), a gatedmicrochannel plate may be used before the diode row for effectiveamplification of the fluorescent light, simultaneously as a suppressionof the background light is obtained.

The detector system will be particularly powerful if, simultaneously asthe spatially resolved signals are registered, also the spectral colourdistribution of the fluorescent light may be obtained. This may be doneby imaging the capillary on the entrance slit of a spectrometer. Theslit is arranged in parallel with the grooves of the grating, whichspectrally separates the light perpendicularly to the slit direction. Ina focal plane the light falls onto a two-dimensional CCD detector, whereone dimension corresponds to different positions along the capillary andcorresponding spectra are in the other dimension. Via a reading unitspectra may be read for the different positions. Spectrometers arrangedin such a way (imaging Spectrometers) are known from satellite-borneremote analysis sensors. A computer may evaluate spectra and comparethem with stored reference spectra for a refined analysis.Alternatively, the intensity in given wavelength ranges may be readcorresponding to the emission bands of fluorescent labels having a knownemission.

A limited spectral analysis may also be obtained by utilising a lineardetector (diode row) if the fluorescent light by means of a specialmirror system is first divided into e.g. four parts which areindividually focused to image lines. By correct positioning, of themirrors these image lines may be placed in a row after each other alongthe linear detector. Into the four separated beam paths other means suchas, band-pass filters are introduced which isolate the fluorescencelight, for example from fluorescent labels used in DNA sequencing. Inthis manner spatially resolved images may simultaneously be obtainedfrom which the positions of the different labels along the capillary maybe seen. The beam division and registering method for fluorescence isperformed here by a technique disclosed in the Swedish patent No. 455646(Fluorescence imaging system).

As mentioned above, the signals may be read and analysed when a suitableseparation between the components has been obtained. Since thecomponents migrate individually at a uniform rate, the information alongthe capillary may, however, be read at several times during a long timeinterval and be co-calculated in the computer to signals exhibiting acombination of favourable signal strength and resolution properties. Inthis way an optimised function as regards speed and resolution may beobtained for an instrument based on the principles given in the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Experimental set-up for separation and analysis of spectralproperties-of sample components.

FIG. 2. Experimental set-up for separation and analysis of spectralproperties of sample components using a cooled CCD camera as a detector.

FIG. 3. Experimental set-up for separation and analysis of spectralproperties of sample components using a spectrometer.

FIG. 4. Experimental set-up for separation and analysis of spectralproperties of sample components using filter means for spectralfiltration of optically divided emitted light.

FIG. 1 shows an experimental set-up with a diode row as detector. Alaser-light source with associated optics (1) irradiates the capillaryelectrophoretic capillary (2). Substances (3) present in the capillary(2) will then fluoresce. The fluorescent light is focused by a lenssystem (4) onto a diode row (5). The signal from the diode row is sentto a computer (6) which on its display screen can show the positions ofthe separated substances (3) in real-time.

FIG. 2 shows an experimental set-up with a cooled CCD camera asdetector. A power unit (1) applies a voltage over a capillary (2) inwhich separation of the sample components takes place. A laser lightsource (3) irradiates the capillary (2) via a modulating lens system(4). The fluorescent light from fluorescing sample components in thecapillary (2) passes via a lens system (5) and a light amplifier (6)into a CCD camera (7) with a cooled detector unit. The signal from theCCD camera is sent to a computer (8) provided with an image processingprogram. The display screen of the computer shows the separation processin the capillary (2) in real-time.

The invention is, of course, not restricted to the embodiments describedabove and shown in the drawings, but many modifications and changes maybe made without departing from the general inventive concept as definedin the subsequent claims.

FIG. 3 shows an experimental set-up with a tow-dimensional CCD detector.A laser-light source with associated optics (1) irradiates the capillaryelectrophoretic capillary (2). Substances (3) present in the capillary(7) will then fluoresce. The fluorescent light is focused by a lenssystem (4) on the entrance slit (9) of a spectrometer (10). Thespectrally resolved light falls on a two-dimensional CCD-detector (5').The signal from the CCD-detector is sent to the computer (6) providedwith an image processing program. The display screen of the computershows the separation process in real time together with the spectralproperties of the sample components (3).

FIG. 4 shows an experimental set-up with a diode row as a detector. Alaser-light source with associated optics (1) irradiates the capillaryelectrophoretic capillary (2). Substances (3) present in the capillary(2) will then fluoresce. The fluorescent light is focused by a lenssystem (4), after which the fluorescent light is split into severalbeams by a mirror system (11) and imaged on a diode row detector (5") inadjacent sections. The beams are individually spectrally filtered byfilters (12), each filter having unique transmission properties. Thesignal from the diode row detector is sent to the computer (6) providedwith an image processing program. The display screen of the computershows the separation process in real time together with the spectralproperties as determined by the filters.

We claim:
 1. A method of analyzing a sample by separating components ofsaid sample in a thin capillary, irradiating said capillary, anddetecting light selected from the group consisting of light emitted byand light absorbed by said components of said sample, comprisingdetecting said light at short intervals during said separatingsimultaneously over the whole or at least a large part of said capillaryto create successive momentary images of the separation pattern andthereby the progress of the separation, thereby permitting terminationof said separating when a desired separation of said components of saidsample is achieved.
 2. The method according to claim 1, wherein saiddetecting is based on fluorescence of said sample components.
 3. Themethod according to claim 1, wherein said detecting is based on lightscattering of said sample components.
 4. The method according to claim1, wherein said irradiating is performed using a laser.
 5. The methodaccording to claim 1, wherein said separating is carried out bycapillary electrophoresis.
 6. The method according to claim 1, 2, 3 or4, wherein said separating is carried out by capillary columnchromatography.
 7. The method according to claim 1, wherein saidseparating is carried out by electrokinetic capillary chromatography. 8.The method of claim 1, wherein the termination of said separating occursprior to complete migration of the sample components through saidcapillary.
 9. The method of claim 1, wherein said irradiating anddetecting occur over substantially the entire length of said capillary.10. The method of claim 1, wherein said irradiating and detecting occurover a majority of the entire length of said capillary.
 11. An apparatusfor sample analysis by monitoring the pattern of separation of samplecomponents and thereby the progress of separation thereof, comprising aseparation capillary, a light source for irradiating said separationcapillary, and a detector for detecting light selected from the groupconsisting of light emitted from and light emitted by said samplecomponents, wherein said light source irradiates the whole or at least alarge part of said separation capillary, and wherein said detector is animaging detector arranged to register data at short intervals during theseparation light emitted from or absorbed by said sample componentsdepending on their spectral characteristics simultaneously over thewhole or at least a large part of said separation capillary to createsuccessive momentary images of said separation pattern and thereby theprogress of said separation.
 12. The apparatus according to claim 11,wherein the imaging detector is a two-dimensional charge coupled device.13. The apparatus according to claim 12, wherein the charge coupleddevice detector is preceded by a spectrometer having an entrance slitonto which said separation capillary containing spatially resolvedsample components is imaged for spectral resolution of light emittedfrom said sample components perpendicularly to said entrance slit, suchthat a spatial resolution is obtained in one direction of thetwo-dimensional detector and a spectral resolution is obtained in theother direction thereof.
 14. The apparatus according to claim 11,wherein said imaging detector is a diode row.
 15. The apparatusaccording to claim 14, wherein said diode row is preceded by (i) a beamdivision system arranged to optically divide said light emitted fromsaid sample components into at least two beam paths and to individuallyfocus said beam paths to image lines placed after one another on saiddiode row, and (ii) filter means for individual spectral filtration ofsaid beam paths of emitted light.
 16. The apparatus according to claim15, wherein said light emitted from said sample components by said beamdivision system is optically divided into four beam paths.
 17. Theapparatus according to any one of claims 11 to 15, which furthercomprises a computer unit arranged to co-process registered data fromsaid imaging detector for characterizing each of said sample componentswith regard to its spectral characteristics and classification bycomparison with spectral information stored in said computer.
 18. Theapparatus of any one of claims 11-15 which further comprises a visualoutput means for displaying registered data.
 19. The apparatus accordingto claim 11, wherein said separation capillary is selected from thegroup consisting of a capillary electrophoresis capillary, anelectrokinetic chromatography capillary, and a capillary for capillarycolumn chromatography.
 20. The apparatus of claim 11, wherein saidimaging detector is more than one diode row.